The Menkes protein (MNK or ATP7A) is a P-type copper transporter ATPase involved in maintaining proper copper homeostasis in cells. It is crucial to maintain intracellular levels of copper for proper cellular function and to prevent the toxic effects excess copper can produce. A disturbed level of copper homeostasis is observed in patients with Menkes disease. This X-linked, multi-system lethal disorder is characterized mental retardation, neurodegeneration, failure to grow, connective tissue disorders and early childhood death. Mutations in MNK can be attributed to the malfunctions of several copper requiring enzymes such as dopamine-B-monooxygenase, cytochrome c oxidase, lysyl oxidase, copperlzinc superoxide dismutase and peptidyiglycine alpha-amidating monooxygenase (PAM). Since PAM is crucial for the amidation of many neuropeptides produced in the brain and pituitary, a reduction in its ability to produce final active products could produce a wide array of biological effects and could contribute to the phenotype of Menkes disease. The first hypothesis to be explored in this proposal is that the endogenous Menkes protein is localized to a vesicular compartment in pituitary endocrine cells. Whole anterior pituitary and dissociated anterior pituitary cell cultures will be used to examine the subcellular localization, biosynthesis and trafficking of the endogenous Menkes protein. Antibodies will be generated to several conserved peptides in the Menkes protein. The second hypothesis I will explore is that the behavior of the pituitary Menkes protein is responsive to copper levels. The copper chelators, bathocuproinedisulfonic acid (BCS) and Disulfiram (Antabuse), will be used to determine if a reduction of copper levels affects the behavior of endogenous MNK. Changes in the levels of amidated peptides produced in the anterior pituitary will be used to evaluate the effectiveness of the drug treatments. My third hypothesis is that the presence of the mutated Menkes protein in the mottled brindled mouse (MObr) will alter pituitary function. Bearing an allelic mutation in the Atp7a gene, the Mobr mice are an excellent model system for examining the consequences of having a mutated Menkes protein. A breeding colony of Mobr mice have been established. The development of the various anterior and intermediate pituitary cell types will be examined, and the expression of mutated MNK in each cell type will be established. Examining the behavior of both the endogenous and mutated Menkes protein in the pituitary, a tissue where the enzyme PAM requires copper for its proper function, will aid our understanding of how copper transporters regulate cellular copper homeostasis. The long-term objective of this proposal is to increase our understanding of how copper transporters provide crucial copper to copper-requiring enzymes that reside in the secretory pathway of cells.